Immunotherapy using tumor-directed chimeric antigen receptor (CAR)-expressing T cells (CART) is a rapidly- emerging, promising cancer therapy that can elicit rapid, durable clinical responses, seen in anti-CD19 CART (CART19)-treated CD19+ B-cell malignancies. However, CART-evoked severe or fatal immune-related adverse events (irAE) including cytokine-release syndrome (CRS) are significant clinical barriers and safety concerns that impede CART use in cancer patients, even with strict patient monitoring and supportive care. Current tools for mitigating such immunotoxicities/CRS target individual cytokines/receptors (e.g. IL-6R, IL-1Ra, GM-CSF) or nonspecific immunosuppression, but preventing and treating such toxicity will require better understanding of cellular/molecular mediators of CART-induced CRS. This project aims to overcome these barriers and enable more effective interventions against immunotoxicities, and thus more effective CART treatments, building upon the PIs' novel, potentially translatable findings of a key role of catecholamines (CATs) in shaping CART therapy- associated immune activation. We found that blocking CAT synthesis both reduced CRS during CART therapy in mice and also enhanced its tumor eradication, and also found that CAT levels are elevated in patients with CART-evoked CRS, and correlated with symptoms and IL-6 levels, suggesting CATs contribute to human CRS pathophysiology. The project's goal is to identify the mechanistic CRS-modulating roles of these novel CAT actions and their impacts on macrophages and anti-tumor responses, and assess translational potential for improving CART cancer therapy outcomes using human patient samples. Specific Aims are: 1) Determine the mediating ?1-AR subtype and its role in modulating CART-evoked cytokine release and anti-tumor responses in vitro and in vivo, based on our finding that CAT-associated cytokine release is mediated by ?1-adrenergic receptors (?1-AR), of as-yet unknown subtype(s). We will identify the ?1-AR subtype(s) that mainly mediate(s) CAT-evoked cytokine production in macrophages and CART, using in vitro co-culture assays and a mouse CART therapy model via genetic knockout (KO) and knockdown (KD) of ?1-AR subtypes. 2) Determine the impact of myeloid-derived CATs on cytokine release and myeloid function during CART-therapy in vitro and in a mouse model, by inactivating CAT synthesis in myeloid cells via tyrosine hydroxylase (TH) KO, and assess its impact on cytokine release and macrophage function in a co-culture assay and in vivo using a LysM-Cre-mediated TH KO mouse. 3) Assess CAT induction in human patients during CART therapy, and CATs' dual impacts on irAE and tumor responses, and potential utility of CAT measures and blockers in predicting and preventing CRS, by quantifying circulating CAT and cytokine levels in patient serum samples collected during CART therapy, and evaluating correlations of CAT levels with clinical and laboratory CRS indices, anti-tumor responses and survival.